information for all tissues utilized is summar-
ized in table S1.
Isolation of cerebrovascular specimens
No differences in vascular isolation methods
were applied between normal cerebral cortex
or AVM tissues. Tissue specimens were placed
in chilled preoxygenated Dulbecco’s modi-
fied Eagle medium (DMEM, Fisher Scientific,
Waltham, MA, catalog number: MT10017CV)
and transported to the laboratory on ice. All
tissue handling was performed with autoclave-
sterilized equipment within a class II biological
safety cabinet. Large arteries and veins were
selectively isolated under 5× magnification
with a Leica MZ75 dissecting microscope
(Leica Microsystems, Wetzlar, Germany) with
two #5/45 Dumont micro-forceps (Fine Science
Tools, Foster City, CA, catalog number: 11251-35).
Under 5X magnification, the lumen of each
vessel was longitudinally opened with a #15
scalpel blade (Fine Science Tools) or flushed
with DMEM. The large vessels were placed
in an Eppendorf LoBind 5-ml tube (Eppendorf
North America, Enfield, CT, catalog number:
0030122348) and kept on ice in chilled oxy-
genated DMEM. Following removal of all vis-
ible vasculature, the microvasculature was
isolated using dextran gradient centrifuga-
tion followed by cell-strainer filtration ( 20 – 22 ).
More specifically, tissue was cut into ~1- to
2-mm pieces with a scalpel and gently homog-
enized in a Dounce homogenizer containing
oxygenated pre-chilled DMEM with 1% bovine
serum albumin (BSA) (Sigma Aldrich, St. Louis,
MO, catalog number: A9418). The homoge-
nate was mixed in 18% dextran solution (MW:
~70,000 Da; Sigma-Aldrich, catalog number:
31390) at a volume ratio of 1:1 and centrifuged
at 6000gfor20minat4°C.Thisresultedina
microvascular pellet and floating vascular-
depleted brain. The floating vessel-depleted
brain was gently aspirated and discarded. The
vascular pellet resuspended in DMEM with
1% BSA and passed through a 40-mm cell
strainer (Fisher Scientific, catalog number:
08-771-1) to remove circulating cells or other
debris. Microvascular fragments remain trapped
on top of the cell strainer and were subsequently
collected by inverting with cell strainer and
washing with prechilled oxygenated DMEM.
A small aliquot was visualized at 10X mag-
nification to confirm both purity and yield
with brightfield microscopy. The microvascu-
lar fragments were pelleted by centrifugation
at 500gfor 5 min. The supernatant was aspi-
rated and then pooled with the microdissected
arteries and veins from the same individual in
prechilled oxygenated DMEM. These pooled
preparations are referred to as isolated vas-
cular preparations for subsequent steps and
maintained in chilled oxygenated media on ice
and immediately processed to create single-cell
suspensions.
Generation of vascular single-cell suspensions
Isolated vascular preparations were incu-
bated for 45 min in 0.2% collagenase type 2
(Worthington Biochemical Corporation,
Lakewood, NJ, catalog number: LS004176)
diluted in preoxygenated DMEM at 37°C with
gentle agitation in an Eppendorf LoBind 5-ml
tube (Eppendorf North America). Cell suspen-
sions were filtered through a sterile 40-mm cell
strainer (Fisher Scientific) to isolate undigested
debris. Cells contained within the flowthrough
were collected by centrifugation at 500gat
5 min and the supernatant carefully aspi-
rated. To lyse any residual erythrocytes, the
cell pellet was resuspended in Gibco ACK
lysing buffer (Fisher Scientific, catalog num-
ber: A1049201) for 3 min at room temperature.
Cells were then collected by centrifugation at
500 gat 5 min and washed three times with
sterile RNase-free phosphate buffered saline
(PBS) (Sigma-Aldrich, catalog number: D8537-
500ml) containing 0.04% BSA. Cells were
pelleted with centrifugation at 500gfor 5 min
and resuspended in PBS with 0.04% BSA. To
confirm cell viability and yield, a 10-ml aliquot
of the cell suspension was mixed 1:1 with 0.4%
trypan blue (Thermo Fisher Scientific, catalog
number: T10282) to stain non-viable cells. Cells
were then counted on a hemocytometer.scRNA-seq
All scRNA-seq experiments were performed
on freshly isolated, whole cells as described
above. Droplet-based scRNA-seq was per-
formed with 10X Genomics Chromium Single
Cell 3 prime reagent kits v3 as described by the
manufacturer (10X Genomics, Pleasanton, CA,
product code: 1000092;n= 5 normal cortex
samples andn=5AVMs).Basedonhemo-
cytometer counts, single cells were loaded
onto chromium chips with a capture target of
15,000 cells per sample. When cell yield was
sufficient, two reactions per individual were
performed. Libraries were prepared follow-
ing the provided manufacturer’s protocol.
Quality of prepared sequencing libraries were
confirmed by electrophoretic analysis on an
Agilent 4200 TapeStation System (Agilent
Technologies, Santa Clara, CA). Libraries were
sequenced with an Illumina NovaSeq 6000
(Illumina, San Diego, CA) with a targeted se-
quencing depth of 50,000 reads per cell.Bulk RNA sequencing
Undigested, isolated vascular tissues from
ruptured and unruptured AVMs were obtained
from the operating room, snap frozen in liquid
nitrogen and stored at−80°C. Snap frozen
tissues were embedded in Optical Cutting
Temperature Compound (Sakura Finetek
USA, Torrance, CA, catalog number: 4583)
and cryosectioned at a thickness of 20mm.
Tissue scrolls were collected in RNAase free
Eppendorf LoBind-1.5 ml microcentrifuge tubes(Eppendorf North America, catalog number:
022431021). DNA/RNA Shield reagent (Zymo
Research, Irvine, CA, catalog number: R1100)
was added. Tissues were mechanically homog-
enized with a Squisher-Single homogenizer
(Zymo Research, catalog number: H1001) and
subsequently digested with proteinase K (Zymo
Research, catalog number: R1057). Total RNA
was isolated from the homogenized tissues with
the Quick-RNA Miniprep Plus Kit as instructed
by the manufacturer (Zymo Research, catalog
number: R1057). The purified RNA was quan-
tified with a NanoDrop (Thermo Fisher Scien-
tific) and integrity confirmed with an Agilent
4200 TapeStation System (Agilent Technolo-
gies, Inc.). Ribosomal RNA was depleted using
the NEBNext rRNA Depletion Kit as instructed
by the manufacturer (New England Biolabs,
Ipswich, MA, catalog number: E6310X), and
sequencing libraries prepared with the SEQuoia
Complete Stranded RNA Library Prep Kit (Bio-
Rad Laboratories, Hercules, CA, catalog num-
ber: 17005710). Sequencing library quality was
confirmed on an Agilent 4200 TapeStation
System (Agilent Technologies, Inc) and quan-
tified with a Qubit dsDNA high sensitivity assay
kit (Thermo Fisher Scientific). Libraries were
sequenced in batch with an Illumina NovaSeq
6000 sequencer (Illumina) with targeted read
depth of at least 5 × 10^7 reads per sample.Rebus Esper spatial omics platform
Spatially resolved, multiplexed in situ RNA de-
tection and analysis was performed using the
automated Rebus Esper spatial omics platform
(Rebus Biosystems, Santa Clara, CA). By inter-
secting a list of known or candidate cell type
markers generated by scRNA-seq, suitability
for probe design, including gene length and
relative abundance, and design constraints for
compatibility with the Rebus Esper spatial
omics platform using proprietary software,
we generated the following gene probe panel:
MECOM,RGS16,RARA,MFSD2A,TAGLN,
IL1R1,VEGFC,KCNJ8,DCN,TMEM132C,
CCL19,CLDN5,PDGFRB,HIGD1B,KCNT2,
ALDH1A2, andALDH1A1.Experiments and
analyses using the Rebus Esper spatial omics
platform were performed as previously de-
scribed ( 70 ).Immunofluorescent staining of tissues and
cerebrovascular fragments
Formalin-fixed paraffin-embedded tissue sec-
tions were cut at a thickness of 6mm, de-
paraffinized with xylene, and rehydrated to
distilled water with serial ethanol washes. For
immunostaining of isolated vessel fragments,
cerebrovascular vessel isolation was performed
as described above and immersion fixed in 4%
paraformaldehyde overnight at 4°C. For anti-
gen retrieval, all specimens were incubated
with pH 9 Tris-EDTA buffer at 97°C for 15 min.
Tissue sections were then blocked with PBSWinkleret al.,Science 375 , eabi7377 (2022) 4 March 2022 9 of 12
RESEARCH | RESEARCH ARTICLE